1. Field of the Invention
This invention relates to a three-dimensional culture containing human articular chondrocytes with induced terminal differentiation changes, as well as the preparation process and uses of said culture.
2. Description of the Related Art
Osteoarthritis (OA) is a joint disease with a gradual degradation of articular cartilage, especially in the aged population (D. Hamerman (1995), Ann Rheum Dis., 54:82-85). Previous reports indicated that 10% of the elderly population (more than 60 years old) suffered from OA in the United States (D. Hamerman (1989), N Engl J Med., 320:1322-1330; G. Peat et al. (2001), Ann Rheum Dis., 60:91-97). Furthermore, a previous report indicated that females with OA mostly suffered from the onset of the symptoms of OA at peri-menopausal stage (M. Y. Nadkar et al. (1999), J Assoc Physicians India, 47:1161-1163). Therefore, it is quite necessary to study the strategies for preventing disease progress at the early stage of OA.
Several previous studies from OA patients indicated that the biological characteristics of articular chondrocytes during the progress of OA included terminal differentiation, mineralization and eventually apoptosis (F. J. Blanco et al. (1998), Arthritis Rheum, 41:284-289; F. Heraud et al. (2000), Ann Rheum Dis, 59:959-965; T. Kirsch et al. (2000), Osteoarthritis Cartilage, 8:294-302). Osteoarthritic chondrocytes were found to express annexin, alkaline phosphatase (ALP) and collagen type X (also called collagen X)(T. Kirsch et al. (2000), Osteoarthritis Cartilage, 8:294-302). Expressions of collagen type X and annexin V reflect the characteristics of hypertrophic chondrocytes as a mature differentiation. Chondrocytic apoptosis was also found in OA cartilage, and it has been suggested that chondrocytic apoptosis might be associated with the decrease of cellularity and abnormal mineralization in OA cartilage (F. J. Blanco et al. (1998), Arthritis Rheum, 41:284-289; S. Hashimoto et al. (1998b), Proc Natl Acad Sci USA, 95:3094-3099; F. Heraud et al. (2000), Ann Rheum Dis, 59:959-965). Accordingly, investigators indicated that the osteoarthritic articular chondrocytes resume the genetic and phenotypic characteristics that were similar to the terminal differentiation of chondrocytes in epiphyseal growth-plates (T. Kirsch et al. (2000), Osteoarthritis Cartilage, 8:294-302). One of the prospects of managing OA is to suppress the terminal differentiation of articular chondrocytes and eventually stop the disease progress in the very early stage of OA. However, studies relevant to an in vitro model applying human articular chondrocytes reaching a terminal differentiation stage for elucidating the progression mechanism of OA have not been reported.
5-azacytidine is a DNA or RNA methyl transferase inhibitor resulting in hypomethylation. It has been reported that 5-azacytidine replaced cytidine in genomic DNA during replication and thus perturbed the methylation pattern of cytidine present in various target gene promoters, such that the transcriptional repression was relieved and the cell differentiation program was changed (J. K. Christman et al. (1983). Cancer Res., 43:763-769; P. A. Jones and S. M. Taylor (1980), Cell, 20:85-93; P. A. Jones et al. (1983), J Exp Zool., 228:287-295; C. Tarella et al. (1982), Cancer Res., 42:445-449; C. Walker et al. (1984), J Natl Cancer Inst., 73:877-885). It was also reported that 5-azacytidine induced the terminal differentiation changes of cultured epiphyseal chondrocytes as occurred during endochondral ossification (J. O. Cheung et al. (2001), J Bone Miner Res., 16:309-318). In addition, M. J. Zuscik et al. established a model to study the regulation mechanism of articular chondrocyte maturation (M. J. Zuscik et al. (2004), J Cell Biochem., 92:316-331). Their study indicated that the 5-azacytidine-treated chicken articular chondrocytes were induced to express the maturational hallmarks, including collagen type X, alkaline phosphatase (ALP), and Indian hedgehog (Ihh), and showed altered collagen type X and alkaline phosphatase expression in response to bone morphogenetic protein-2 (BMP-2), transforming growth factor-β (TGF-β), and parathyroid hormone-related protein (PTHrP). However, this model has yet to be applied to cultured human articular chondrocytes.
Alginate is a copolymer of L-guluronic acid and D-mannuronic acid that polymerizes to form a gel in the presence of calcium ions. Alginate can easily be depolymerized with the addition of a calcium chelator such as sodium citrate or EDTA. Alginate has found application in cell encapsulation, cell transplantation, and tissue engineering. Cells that are suspended in an alginate solution may be entrapped in alginate beads with three-dimensional configuration that form upon polymerization. These alginate beads can be used for in vitro culture and also for transplantation in vivo into articular cartilage defects. Previous studies using human articular chondrocytes cultured in alginate beads have shown that these human articular chondrocytes secrete a matrix similar to that seen in native human cartilage, and maintain their phenotype over long periods of time.
Based on the findings from the aforesaid studies, the applicants endeavored to develop a three-dimensional culture containing human articular chondrocytes that may be induced to reach a terminal differentiation stage, so that the three-dimensional culture containing human articular chondrocytes can act as a cell culture model mimicking the terminal differentiation occurring in osteoarthritic chondrocytes, can be used for identifying a pathology associated with articular chondrocytes, such as osteoarthritis, and can be used for screening a candidate drug in the treatment of a disorder associated with articular chondrocytes, such as osteoarthritis.